1. Field of the Invention
This invention relates to variants of cytochrome P450 oxygenases. Specifically, the invention relates to thermostable variants of cytochrome P450 oxygenases capable of improved peroxide-driven hydroxylation, and methods of making thermostable variants.
2. Background Information
One of the great challenges of contemporary catalysis is the controlled oxidation of hydrocarbons. Processes for controlled, stereo- and regioselective oxidation of hydrocarbon feed stocks to more valuable and useful products such as alcohols, ketones, acids, and peroxides would have a major impact on the chemical and pharmaceutical industries. However, selective oxyfunctionalization of hydrocarbons remains one of the great challenges for contemporary chemistry. Despite decades of effort, including recent advances, the insertion of oxygen into unactivated carbon-hydrogen bonds (hydroxylation) remains difficult to achieve with high selectivity and high yield. Many chemical methods for hydroxylation require severe conditions of temperature or pressure, and the reactions are prone to over-oxidation, producing a range of products, many of which are not desired.
Enzymes are an attractive alternative to chemical catalysts. In particular, mono-oxygenases have unique properties that distinguish them from most chemical catalysts. Most impressive is their ability to catalyze the specific hydroxylation of non-activated C—H, one of the most useful biotransformation reactions, which is often difficult to achieve by chemical means, especially in water, at room temperature and atmospheric pressure. These cofactor-dependent oxidative enzymes have multiple domains and function via complex electron transfer mechanisms to transport a reduction equivalent to the catalytic heme center.
Cytochrome P450 monooxygenases (“P450s”) are a group of widely-distributed heme-containing enzymes that insert one oxygen atom from diatomic oxygen into a diverse range of hydrophobic substrates, often with high regio- and stereoselectivity. The second oxygen atom is reduced to H2O. The active sites of all cytochrome P450s contain an iron protoporphyrin IX with cysteinate as the fifth ligand, and the final coordination site is left to bind and activate molecular oxygen. Their ability to catalyze these reactions with high specificity and selectivity makes P450s attractive catalysts for chemical synthesis and other applications, including oxidation chemistry, and for many of the P450-catalyzed reactions, no chemical catalysts come close in performance. These enzymes are able to selectively hydroxylate a wide range of compounds, including fatty acids, aromatic compounds, alkanes, alkenes, and natural products. Unfortunately, P450s are generally limited by low turnover rates, and they generally require an expensive cofactor, NADH or NADPH, and at least one electron transfer partner protein (reductase). Furthermore, the enzymes are large, complex, and expensive.
Wild-type P450s are in some cases capable of using peroxides as a source of oxygen and electrons via a peroxide “shunt” pathway, though the efficiency of this route is low. This secondary mechanism for substrate oxidation offers the opportunity to take advantage of P450 catalysis without the need for a cofactor, and eliminates the rate-limiting electron transfer step carried out by the reductase. However, low efficiency is a major limitation. Further, wild-type enzymes capable of peroxide-driven hydroxylation, such as chloroperoxidase (CPO) and CYP152B1 are generally limited in their substrate specificity to hydroxylation of activated C—H bond carbons, i.e., carbon atoms adjacent to a functional group such as an aromatic ring, a carbonyl group, a heteroatom, etc.
One particular P450 enzyme, cytochrome P450 BM-3 from Bacillus megaterium (“P450 BM-3”; EC 1.14.14.1) also known as CYP102, is a water-soluble, catalytically self-sufficient P450 containing a heme (monooxygenase/hydroxylase) domain which is 472 amino acids in length and a reductase domain that is 585 amino acids in length. The total length of the enzyme is 1048 amino acids. The heme domain is generally considered to end at position 472 and it is followed by a short linker before the reductase domain begins. Because of the presence of an independent reductase domain within the protein itself, P450 BM-3 does not require an additional or extraneous reductase for activity, but it does require an electron source, such as the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). Nucleotide and amino acid sequences for P450 BM-3 are provided in FIGS. 1 and 2, respectively, which are the sequences for P450 BM-3 from the GenBank database, accession nos. J04832 (SEQ ID NO:1) and P14779 (SEQ ID NO:2), respectively.
P450 BM-3 hydroxylates fatty acids with a chain length between C12 and C18 at subterminal positions, and the regioselectivity of oxygen insertion depends on the chain length. The optimal chain length of saturated fatty acids for P450 BM-3 is 14-16 carbons. P450 BM-3 is also known to hydroxylate the corresponding fatty acid amides and alcohols and forms epoxides from unsaturated fatty acids. The minimum requirements for activity are substrate, diatomic oxygen, and the cofactor NADPH.
It has been demonstrated that ω-para-nitrophenoxycarboxylic acids (pNCAs) can be used as surrogate substrates for BM-3. When this substrate is hydroxylated at the ω position to produce ω-oxycarboxylic acid, the yellow chromophore p-nitrophenolate (pNP) is produced, allowing for easy detection of activity when screening mutant libraries.
Mutant P450 BM-3 enzymes with modified activity have now been reported in the literature. For example, an F87A mutant was found to display a higher activity for the 12-pNCA substrate, and, under NADPH-driven catalysis, resulted in complete terminal hydroxylation of 12-pNCA, whereas the wild-type enzyme stopped at about 33% conversion. It has also been reported that the F87A mutant has a higher stability in H2O2 solutions. (The convention in the art, which is adopted herein, is to refer to a mutant with reference to the native amino acid residue at a position in the sequence, followed by the amino acid at that position in the mutant, e.g., F87 refers to the phenylalanine at position 87 in the wild-type sequence, and F87A refers to the phenylalanine at position 87 in the wild-type sequence which has been changed to alanine in the variant. The numbering of the amino acid residues starts with the amino acid residue following the initial methionine residue). It has been shown that H2O2-driven hydroxylation to be much faster with the F87A mutation, as well as with an F87G mutation.
Powerful techniques for creating enzymes with modified or improved properties are now available, such as directed evolution (Arnold, 1998), in which iterative cycles of random mutagenesis, recombination and functional screening for improved enzymes accumulate the mutations that confer the desired properties. For example, mutants of cytochrome P450cam or P450 BM-3 that hydroxylate the activated C—H bonds of naphthalene or 12-pNCA substrate, respectively, in the absence of co-factors through the “peroxide-shunt” pathway, herein termed “peroxygenases,” have been created and identified using such techniques. In addition, P450 BM-3 mutants that can hydroxylate a variety of nonnatural substrates, including octane, several aromatic compounds and heterocyclic compounds have been reported.
While the activity of enzymes has thus been improved and modified, a continuing problem is that enzymes are often poorly stable under conditions encountered during production, storage or use. For example, improving enzyme resistance to thermal denaturation has been a major focus of protein engineering efforts. Improved thermostability often correlates with longer shelf-life, longer life-time during use (even at low temperatures), and a higher temperature optimum for activity. Stabilizing the relatively unstable cytochrome P450 enzymes by protein engineering is a particularly challenging problem, however, partly because the P450s comprise multiple subunits and contain thermolabile co-factors. Two thermostable cytochrome P450s (CYP119 and CYP175A1) from thermophilic organisms have recently been described and their (heme domain) crystal structures determined. CYP119 exhibits a melting temperature of ˜91° C. Aromatic stacking, salt-link networks and shortened loops are believed to help stabilize these enzymes. Unfortunately, the functions of these P450s are not known, and reported activities are low (e.g., 0.35 min−1 in the NADH-driven hydroxylation of lauric acid. While the International Patent application published as WO 02/083868 found that the mutations M145A, L324I, I366V, and E442K in the P450 BM-3 heme domain promoted thermostability, the overall thermostability of the peroxygenase mutant was not higher than that of the wild-type heme domain.
Thus, there is a need in the art for useful oxidation catalysts which are stable and do not require expensive cofactors or coenzymes for efficient oxidation and for methods of preparing the same. This invention addresses these and other needs in the art.